The present invention relates to the potentiation of glutamate receptor function using certain heterocyclyl sulphonamide derivatives. It also relates to novel heterocyclyl sulphonamide derivatives, to processes for their preparation and to pharmaceutical compositions containing them.
In the mammalian central nervous system (CNS), the transmission of nerve impulses is controlled by the interaction between a neurotransmitter, that is released by a sending neuron, and a surface receptor on a receiving neuron, which causes excitation of this receiving neuron. L-Glutamate, which is the most abundant neurotransmitter in the CNS, mediates the major excitatory pathway in mammals, and is referred to as an excitatory amino acid (EAA). The receptors that respond to glutamate are called excitatory amino acid receptors (EAA receptors). See Watkins and Evans, Ann. Rev. Pharmacol. Toxicol., 21, 165 (1981); Monaghan, Bridges, and Cotman, Ann. Rev. Pharmacol. Toxicol., 29, 365 (1989); Watkins, Krogsgaard-Larsen, and Honore, Trans. Pharm. Sci., 11, 25 (1990). The excitatory amino acids are of great physiological importance, playing a role in a variety of physiological processes, such as long-term potentiation (learning and memory), the development of synaptic plasticity, motor control, respiration, cardiovascular regulation, and sensory perception.
Excitatory amino acid receptors are classified into two general types. Receptors that are directly coupled to the opening of cation channels in the cell membrane of the neurons are termed xe2x80x9cionotropicxe2x80x9d. This type of receptor has been subdivided into at least three subtypes, which are defined by the depolarizing actions of the selective agonists N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), and kainic acid (KA). The second general type of receptor is the G-protein or second messenger-linked xe2x80x9cmetabotropicxe2x80x9d excitatory amino acid receptor. This second type is coupled to multiple second messenger systems that lead to enhanced phosphoinositide hydrolysis, activation of phospholipase D, increases or decreases in c-AMP formation, and changes in ion channel function. Schoepp and Conn, Trends in Pharmacol. Sci.,14, 13 (1993). Both types of receptors appear not only to mediate normal synaptic transmission along excitatory pathways, but also participate in the modification of synaptic connections during development and throughout life. Schoepp, Bockaert, and Sladeczek, Trends in Pharmacol. Sci.,11, 508 (1990); McDonald and Johnson, Brain Research Reviews, 15, 41 (1990).
AMPA receptors are assembled from four protein sub-units known as GluR1 to GluR4, while kainic acid receptors are assembled from the sub-units GluR5 to GluR7, and KA-1 and KA-2. Wong and Mayer, Molecular Pharmacology 44: 505-510, 1993. It is not yet known how these sub-units are combined in the natural state. However, the structures of certain human variants of each sub-unit have been elucidated, and cell lines expressing individual sub-unit variants have been cloned and incorporated into test systems designed to identify compounds which bind to or interact with them, and hence which may modulate their function. Thus, European patent application, publication number EP-A2-0574257 discloses the human sub-unit variants GluR1B, GluR2B, GluR3A and GluR3B. European patent application, publication number EP-A1-0583917 discloses the human sub-unit variant GluR4B.
One distinctive property of AMPA and kainic acid receptors is their rapid deactivation and desensitization to glutamate. Yamada and Tang, The Journal of Neuroscience, September 1993, 13(9): 3904-3915 and Kathryn M. Partin, J. Neuroscience, Nov. 1, 1996, 16(21): 6634-6647. The physiological implications of rapid desensitization, and deactivation if any, are unknown.
It is known that the rapid desensitization and deactivation of AMPA and/or kainic acid receptors to glutamate may be inhibited using certain compounds. This action of these compounds is often referred to in the alternative as xe2x80x9cpotentiationxe2x80x9d of the receptors. One such compound, which selectively potentiates AMPA receptor function, is cyclothiazide. Partin et al., Neuron. Vol. 11, 1069-1082, 1993. Compounds which potentiate AMPA receptors, like cyclothiazide, are often referred to as ampakines.
International Patent Application Publication Number WO 9625926 discloses a group of phenylthioalkylsulphonamides, S-oxides and homologs which are said to potentiate membrane currents induced by kainic acid and AMPA.
Ampakines have been shown to improve memory in a variety of animal tests. Staubli et al., Proc. Natl. Acad. Sci., Vol. 91, pp 777-781, 1994, Neurobiology, and Arai et al., The Journal of Pharmacology and Experimental Therapeutics, 278: 627-638, 1996.
It has now been found that cyclothiazide and certain heterocyclyl sulphonamide derivatives potentiate agonist-induced excitability of human GluR4B receptor expressed in HEK 293 cells. Since cyclothiazide is known to potentiate glutamate receptor function in vivo, it is believed that this finding portends that the sulphonamide derivatives will also potentiate glutamate receptor function in vivo, and hence that the compounds will exhibit ampakine-like behavior.
Accordingly, the present invention provides a compound of the formula: 
wherein:
R1 represents a carbon-linked 4 to 7 membered saturated heterocyclic ring containing as the hetero ring members a group NRa and a group Xa in which Xa represents CH2, CO, O, S or NRb; Ra represents hydrogen or (1-4C)alkyl; and Rb represents hydrogen or (1-4C)alkyl;
L represents a bond or a group of formula xe2x80x94(La)mxe2x80x94Xxe2x80x94(Lb)nxe2x80x94in which X represents O, S, NRc, CONH or NHCO; La represents (1-4C)alkylene; Lb represents (1-4C)alkylene; m is zero or 1; n is zero or 1; and Rc is hydrogen or (1-4C)alkyl;
R2 represents (1-6C)alkyl, (3-6C)cycloalkyl, (1-6C)fluoroalkyl, (1-6C)chloroalkyl, (2-6C)alkenyl, (1-4C)alkoxy(1-4C)alkyl, phenyl which is unsubstituted or substituted by halogen, (1-4C)alkyl or (1-4C)alkoxy, or a group of formula R3R4N in which R3 and R4 each independently represents (1-4C)alkyl or, together with the nitrogen atom to which they are attached form an azetidinyl, pyrrolidinyl, piperidinyl, morpholino, piperazinyl, hexahydroazepinyl or octahydroazocinyl group; and
either (a) one or two of R5, R6, R7 and R8 represents hydrogen; (1-6C)alkyl; aryl(1-6C)alkyl; (2-6C)alkenyl; aryl(2-6C)alkenyl or aryl, or (b) two of R5, R6, R7 and R8 together with the carbon atom or carbon atoms to which they are attached form a (3-8C) carbocyclic ring; and the remainder of R5, R6, R7 and R8 represent hydrogen; or a pharmaceutically acceptable salt thereof, provided that when L represents a group of formula xe2x80x94(La)mxe2x80x94Xxe2x80x94(Lb)n and m is 0, R1 is not pyrrolidinyl.
According to another aspect, the present invention provides a method of potentiating glutamate receptor function in a mammal (including a human) requiring such treatment, which comprises administering an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof as defined herein.
According to another aspect, the present invention provides the use of a compound of formula I, or a pharmaceutically acceptable salt thereof as defined herein for the manufacture of a medicament for potentiating glutamate receptor function.
According to yet another aspect, the present invention provides the use of a compound of formula I or a pharmaceutically acceptable salt thereof as defined herein for potentiating glutamate receptor function.
In this specification, the term xe2x80x9cpotentiating glutamate receptor functionxe2x80x9d refers to any increased responsiveness of glutamate receptors, for example AMPA receptors, to glutamate or an agonist, and includes but is not limited to inhibition of rapid desensitisation or deactivation of AMPA receptors to glutamate.
A wide variety of conditions may be treated by the compounds of formula I and their pharmaceutically acceptable salts through their action as potentiators of glutamate receptor function. Such conditions include those associated with glutamate hypofunction, such as psychiatric and neurological disorders, for example cognitive disorders; neuro-degenerative disorders such as Alzheimer""s disease; age-related dementias; age-induced memory impairment; movement disorders such as tardive dyskinesia, Hungtington""s chorea, myoclonus and Parkinson""s disease; reversal of drug-induced states (such as cocaine, amphetamines, alcohol-induced states); depression; attention deficit disorder; attention deficit hyperactivity disorder; psychosis;
cognitive deficits associated with psychosis; and drug-induced psychosis. The compounds of formula I may also be useful for improving memory (both short term and long term) and learning ability. The present invention provides the use of compounds of formula I for the treatment of each of these conditions.
The term xe2x80x9ctreatingxe2x80x9d (or xe2x80x9ctreatxe2x80x9d) as used herein includes its generally accepted meaning which encompasses prohibiting, preventing, restraining, and slowing, stopping, or reversing progression, severity, or a resultant symptom.
The present invention includes the pharmaceutically acceptable salts of the compounds defined by formula I. A compound of this invention can possess a sufficiently acidic, a sufficiently basic, or both functional groups, and accordingly react with any of a number of organic and inorganic bases, and inorganic and organic acids, to form a pharmaceutically acceptable salt.
The term xe2x80x9cpharmaceutically acceptable saltxe2x80x9d as used herein, refers to salts of the compounds of the above formula which are substantially non-toxic to living organisms. Typical pharmaceutically acceptable salts include those salts prepared by reaction of the compounds of the present invention with a pharmaceutically acceptable mineral or organic acid or an organic or inorganic base. Such salts are known as acid addition and base addition salts.
Acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of such pharmaceutically acceptable salts are the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, hydrochloride, dihydrochloride, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate, phthalate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, xcex3-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, napththalene-2-sulfonate, mandelate and the like. Preferred pharmaceutically acceptable acid addition salts are those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and those formed with organic acids such as maleic acid and methanesulfonic acid.
Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Such bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like. The potassium and sodium salt forms are particularly preferred. It should be recognized that the particular counterion forming a part of any salt of this invention is usually not of a critical nature, so long as the salt as a whole is pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole. It is further understood that the above salts may form hydrates or exist in a substantially anhydrous form.
As used herein, the term xe2x80x9cstereoisomerxe2x80x9d refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures which are not interchangeable. The three-dimensional structures are called configurations. As used herein, the term xe2x80x9cenantiomerxe2x80x9d refers to two stereoisomers whose molecules are nonsuperimposable mirror images of one another. The term xe2x80x9cchiral centerxe2x80x9d refers to a carbon atom to which four different groups are attached. As used herein, the term xe2x80x9cdiastereomersxe2x80x9d refers to stereoisomers which are not enantiomers. In addition, two diastereomers which have a different configuration at only one chiral center are referred to herein as xe2x80x9cepimersxe2x80x9d. The terms xe2x80x9cracematexe2x80x9d, xe2x80x9cracemic mixturexe2x80x9d or xe2x80x9cracemic modificationxe2x80x9d refer to a mixture of equal parts of enantiomers.
The term xe2x80x9cenantiomeric enrichmentxe2x80x9d as used herein refers to the increase in the amount of one enantiomer as compared to the other. A convenient method of expressing the enantiomeric enrichment achieved is the concept of enantiomeric excess, or xe2x80x9ceexe2x80x9d, which is found using the following equation:   ee  =                              E          1                -                  E          2                                      E          1                +                  E          2                      xc3x97    100  
wherein E1 is the amount of the first enantiomer and E2 is the amount of the second enantiomer. Thus, if the initial ratio of the two enantiomers is 50:50, such as is present in a racemic mixture, and an enantiomeric enrichment sufficient to produce a final ratio of 50:30 is achieved, the ee with respect to the first enantiomer is 25%. However, if the final ratio is 90:10, the ee with respect to the first enantiomer is 80%. An ee of greater than 90% is preferred, an ee of greater than 95% is most preferred and an ee of greater than 99% is most especially preferred. Enantiomeric enrichment is readily determined by one of ordinary skill in the art using standard techniques and procedures, such as gas or high performance liquid chromatography with a chiral column. Choice of the appropriate chiral column, eluent and conditions necessary to effect separation of the enantiomeric pair is well within the knowledge of one of ordinary skill in the art. In addition, the enantiomers of compounds of formula I can be resolved by one of ordinary skill in the art using standard techniques well known in the art, such as those described by J. Jacques, et al., xe2x80x9cEnantiomers, Racemates, and Resolutionsxe2x80x9d, John Wiley and Sons, Inc., 1981. Examples of resolutions include recrystallization techniques or chiral chromatography.
Some of the compounds of the present invention have one or more chiral centers and may exist in a variety of stereoisomeric configurations. As a consequence of these chiral centers, the compounds of the present invention occur as racemates, mixtures of enantiomers and as individual enantiomers, as well as diastereomers and mixtures of diastereomers. All such racemates, enantiomers, and diastereomers are within the scope of the present invention.
The terms xe2x80x9cRxe2x80x9d and xe2x80x9cSxe2x80x9d are used herein as commonly used in organic chemistry to denote specific configuration of a chiral center. The term xe2x80x9cRxe2x80x9d (rectus) refers to that configuration of a chiral center with a clockwise relationship of group priorities (highest to second lowest) when viewed along the bond toward the lowest priority group. The term xe2x80x9cSxe2x80x9d (sinister) refers to that configuration of a chiral center with a counterclockwise relationship of group priorities (highest to second lowest) when viewed along the bond toward the lowest priority group. The priority of groups is based upon their atomic number (in order of decreasing atomic number). A partial list of priorities and a discussion of stereochemistry is contained in xe2x80x9cNomenclature of Organic Compounds: Principles and Practicexe2x80x9d, (J. H. Fletcher, et al., eds., 1974) at pages 103-120.
Examples of values for a carbon-linked 4 to 7 membered saturated heterocyclic ring are azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, hexahydropyrimidyl, tetrahydro-1,3-oxazinyl, tetrahydro-1,3-thiazinyl and hexahydroazepinyl.
Examples of more particular values for a carbon-linked 4 to 7 membered saturated heterocyclic ring are: 
wherein Ra represents hydrogen or (1-4C)alkyl; and Rb represents hydrogen or (1-4C)alkyl
Examples of values for Ra are hydrogen and methyl.
Examples of values for Rb are hydrogen and methyl.
Examples of values for Rc are hydrogen and methyl.
Examples of values for Xa are CH2 and CO.
As used herein, the xe2x80x9carylxe2x80x9d as such or in the term xe2x80x9caryl(1-6C)alkylxe2x80x9d means phenyl or a polycyclic aromatic carbocyclic ring such as naphthyl.
The term (1-6C)alkyl includes (1-4C)alkyl. Examples of particular values are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl and hexyl.
Examples of particular values for the term (2-6C)alkenyl are vinyl and prop-2-enyl.
The term (3-8C)cycloalkyl, as such or in the term (3-8C)cycloalkyloxy, includes monocyclic and polycyclic groups. Examples of particular values are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and bicyclo[2.2.2]octane.
The term halogen includes fluorine, chlorine, bromine and iodine.
Examples of particular values for the term fluoro(1-6C)alkyl are trifluoromethyl and 2,2,2-trifluoroethyl, and for chloro(1-6C)alkyl, chloromethyl.
Examples of particular values for the term (1-4C)alkoxy are methoxy and ethoxy.
An example of a particular value for the term (1-4C)alkoxy(1-4C)alkoxy is methoxymethyl.
Examples of particular values for the term (1-20 4C)alkylene are methylene, ethylene, propylene and butylene.
Examples of particular values for R1 are 3-piperidinyl, 4-piperidinyl, 6-oxo-3-piperidinyl, 2-oxo-4-piperidinyl, N-methyl-3-piperidinyl and N-methyl-4-piperidinyl.
Examples of particular values for La are methylene, ethylene, propylene and butylene.
Examples of particular values for Lb are methylene, ethylene, propylene and butylene.
An example of a particular value for m is 1.
An example of a particular value for n is 0.
An example of a particular value for X is 0.
L preferably represents a bond or a group of formula LaX.
Examples of values for R2 are methyl, ethyl, propyl, 2-propyl, butyl, 2-methylpropyl, cyclohexyl, trifluoromethyl, 2,2,2-trifluoroethyl, chloromethyl, ethenyl, prop-2-enyl, ethoxyethyl, phenyl, 4-fluorophenyl, or dimethylamino. Preferably R2 is ethyl, 2-propyl or dimethylamino.
Preferably R3 and R4 each represent methyl.
Examples of a (1-6C)alkyl group represented by R5, R6, R7 and R8 are methyl, ethyl and propyl. An example of an aryl(1-C)alkyl group is benzyl. An example of a (2-6C)alkenyl group is prop-2-enyl. An example of a (3-8C)carbocyclic ring is a cyclopropyl ring.
Preferably R6 and R7 each represents hydrogen.
Preferably R5 and R8 each independently represents hydrogen or (1-4C)alkyl, or together with the carbon atom to which they are attached form a (3-8C) carbocyclic ring.
More preferably R8 represents methyl or ethyl and R5 represents hydrogen or methyl, or R5 and R8 together with the carbon atom to which they are attached form a cyclopropyl ring.
Especially preferred are compounds in which R8 represents methyl and R5, R6 and R7 represent hydrogen.
The compounds according to the invention may be prepared by:
(a) reacting a compound of formula 
xe2x80x83with a compound of formula
R2SO2Z1xe2x80x83xe2x80x83III
xe2x80x83in which Z1 represents a leaving atom or group;
(b) for a compound of formula I in which L represents a bond and R1 represents a 6-oxo-3-piperidinyl group or a 2-oxo-4-piperidinyl group, rearranging a compound of formula 
(c) for a compound of formula I in which L represents xe2x80x94(La)mxe2x80x94Xxe2x80x94(Lb)nxe2x80x94 and X represents O, S or NRc, reacting a compound of formula 
xe2x80x83with a compound of formula
R1xe2x80x94(La)mxe2x80x94Z2xe2x80x83xe2x80x83VI
xe2x80x83in which Z2 represents a leaving atom or group,
(d) for a compound of formula I in which L represents xe2x80x94(La)mxe2x80x94Xxe2x80x94(Lb)nxe2x80x94 and X represents CONH or NHCO, reacting a compound of formula 
xe2x80x83with a compound of formula
R1xe2x80x94(La)mxe2x80x94Mbxe2x80x83xe2x80x83VIII
xe2x80x83in which one of Ma and Mb represents an amino group and the other represents a carboxyl group or a reactive derivative thereof; or
(e) for a compound of formula I in which L represents a bond reacting a compound of formula 
xe2x80x83in which Z3 represents a halogen atom, with a borane of formula
R1BRxRyxe2x80x83xe2x80x83X
xe2x80x83in which each of Rx and Ry represents a hydrocarbyl group, or together represents a hydrocarbyl group; followed, if desired, by forming a pharmaceutically acceptable salt thereof.
In step (a) of the process, the leaving atom or group represented by Z1 may be, for example, a halogen atom such as a chlorine or bromine atom. The reaction is conveniently performed in the presence of a base, for example an alkali metal hydroxide such as sodium hydroxide, an alkali metal carbonate such as potassium carbonate, a tertiary amine such as triethylamine or 1,8-diazabicyclo[5.4.0]undec-7-ene. Suitable solvents include halogenated hydrocarbons such as dichloromethane. The reaction is conveniently performed at a temperature in the range of from xe2x88x9220 to 100xc2x0 C., preferably from xe2x88x925 to 50xc2x0 C.
Step (b) of the process represents an application of the well known Beckmann rearrangement. The rearrangement is conveniently performed in the presence of an organosulfonyl-oxyhalide, such as p-toluenesulfonyl chloride in the presence of a base, for example an alkali metal hydroxide such as sodium hydroxide. Convenient solvents include aqueous ethers, such as aqueous dioxane. The temperature is conveniently in the range of from xe2x88x9210 to 50xc2x0 C. The 6-oxo-3-piperidinyl group or 2-oxo-4-piperidinyl group may then, if desired, be reduced to afford respectively a 3-piperidinyl or 4-piperidinyl group, for example using a borane, such as borane-tetrahydrofuran complex. Convenient solvents include ethers such as tetrahydrofuran. A 3-piperidinyl or 4-piperidinyl group may then, if desired, be alkylated, for example by reductive amination. For example, the group may be methylated using formic acid and formaldehyde.
In step (c) of the process, the leaving atom or group represented by Z2 may be, for example, a halogen atom such as a chlorine or bromine atom. The reaction is conveniently performed in the presence of a base, for example an alkali metal hydroxide such as sodium hydroxide, an alkali metal carbonate such as potassium carbonate, a tertiary amine such as triethylamine or 1,8-diazabicyclo[5.4.0]undec-7-ene. Suitable solvents include halogenated hydrocarbons such as dichloromethane. The reaction is conveniently performed at a temperature in the range of from xe2x88x9220 to 100xc2x0 C., preferably from xe2x88x925 to 50xc2x0 C.
Step (d) of the process corresponds with the well known acylation of an amine. Examples of a reactive derivative of a carboxyl group thus include acyl halides, such as the chloride, and anhydrides.
The halogen atom represented by Z3 in step (e) of the process may be, for example, a bromine atom. In the borane, the groups Rx and Ry may together represent, for example, cyclo-1,5-octyl. The reaction is conveniently performed in the presence of a tetrakis (triarylphosphine)palladium(0) catalyst, such as tetrakis (triphenylphosphine)palladium(0), and a base, such as potassium carbonate or potassium phosphate. Convenient solvents for the reaction include aromatic hydrocarbons, such as toluene. The temperature is conveniently in the range of from 0 to 150xc2x0 C., preferably from 75 to 120xc2x0 C. The borane starting material may be prepared by reacting a corresponding carbon-linked 4 to 7 membered monounsaturated heterocyclic compound, such as 2,5-dihydropyrrole, with an appropriate organoborane, such as 9-borabicyclo[3.3.1]nonane (9-BBN).
The compounds of formula II are known or may be prepared by conventional methods, for example by reducing a corresponding amide or nitrile using borane.
The compounds of formula IV may be prepared by reacting a compound of formula 
with hydroxylamine or an acid addition salt thereof.
The compounds of formula XI may be prepared by reducing a compound of formula 
for example by catalytic hydrogenation in the presence of a Group VIII metal catalyst, such as palladium on carbon.
The compounds of formula XII may be prepared by reacting a compound of formula IX with 3-tributyltin-2-cyclopenten-1-one in the presence of dichlorobis(triphenyl-phosphine)palladium(II). Convenient solvents include ethers such as tetrahydrofuran. The temperature is conveniently in the range of from 0 to 100xc2x0 C. The tributyltin compound may be prepared by reacting hexabutylditin with butyl lithium, followed by 3-ethoxy-2-cyclopenten-1-one at a reduced temperature.
The compounds of formula IX may be prepared by reacting a compound of formula XIII 
with a compound of formula III according to the method of process (a) above.
The compounds of formula V may be prepared by reacting a compound of formula 
or a derivative thereof substituted on X with a protecting group, for example a benzyl group, with a compound of formula III, according to the method of step (a) above. A benzyl protecting group may be removed, for example, by reaction with ammonium formate in the presence of palladium on carbon. A t-butoxycarbonyl nitrogen protecting group may be introduced, for example, by reaction of an unprotected compound with di-tert-butyl dicarbonate, conveniently in the presence of a base such as 4-dimethylaminopyridine. Suitable solvents include halogenated hydrocarbons, such as dichloromethane.
The compounds of formula VII may be prepared by a method analogous to that used to prepare the compounds of formula V.
The compounds of formula XIII and XIV are known or may be prepared by conventional methods, for example by reducing a corresponding amide or nitrile using borane.
The ability of compounds of formula I to potentiate glutamate receptor-mediated response may be determined using fluorescent calcium indicator dyes (Molecular Probes, Eugene, Oreg., Fluo-3) and by measuring glutamate-evoked efflux of calcium into GluR4 transfected HEK293 cells, as described in more detail below.
In one test, 96 well plates containing confluent monolayers of HEK cells stably expressing human GluR4B (obtained as described in European Patent Application Publication Number EP-A1-583917) are prepared. The tissue culture medium in the wells is then discarded, and the wells are each washed once with 200 xcexcl of buffer (glucose, 10 mM, sodium chloride, 138 mM, magnesium chloride, 1 mM, potassium chloride, 5 mM, calcium chloride, 5 mM, N-[2-hydroxyethyl]-piperazine-N-[2-ethanesulfonic acid], 10 mM, to pH 7.1 to 7.3). The plates are then incubated for 60 minutes in the dark with 20 xcexcM Fluo3-AM dye (obtained from Molecular Probes Inc., Eugene, Oreg.) in buffer in each well. After the incubation, each well is washed once with 100 xcexcl buffer, 200 xcexcl of buffer is added and the plates are incubated for 30 minutes.
Solutions for use in the test are also prepared as follows. 30 xcexcM, 10 xcexcM, 3 xcexcM and 1 xcexcM dilutions of test compound are prepared using buffer from a 10 mM solution of test compound in DMSO. 100 xcexcM cyclothiazide solution is prepared by adding 3 xcexcl of 100 mM cyclothiazide to 3 ml of buffer. Control buffer solution is prepared by adding 1.5 xcexcl DMSO to 498.5 xcexcl of buffer.
Each test is then performed as follows. 200 xcexcl of control buffer in each well is discarded and replaced with 45 xcexcl of control buffer solution. A baseline fluorescent measurement is taken using a FLUOROSKAN II fluorimeter (Obtained from Labsystems, Needham Heights, Mass., USA, a Division of Life Sciences International Plc). The buffer is then removed and replaced with 45 xcexcl of buffer and 45 xcexcl of test compound in buffer in appropriate wells. A second fluorescent reading is taken after 5 minutes incubation. 15 xcexcl of 400 xcexcM glutamate solution is then added to each well (final glutamate concentration 100 4 xcexcM), and a third reading is taken. The activities of test compounds and cyclothiazide solutions are determined by subtracting the second from the third reading (fluorescence due to addition of glutamate in the presence or absence of test compound or cyclothiazide) and are expressed relative to enhance fluorescence produced by 100 xcexcM cyclothiazide.
In another test, HEK293 cells stably expressing human GluR4 (obtained as described in European Patent Application Publication No. EP-A1-0583917) are used in the electro-physiological characterization of AMPA receptor potentiators. The extracellular recording solution contains (in mM): 140 NaCl, 5 KCl, 10 HEPES, 1 MgCl2, 2 CaCl2, 10 glucose, pH=7.4 with NaOH, 295 mOsm kgxe2x88x921. The intracellular recording solution contains (in nM): 140 CsCl, 1 MgCl2, 10 HEPES, (N-[2-hydroxyethyl]piperazine-N1-[2-ethanesulfonic acid]) 10 EGTA (ethylene-bis(oxyethylenenitrilo) tetraacetic acid), pH=7.2 with CsOH, 295 mOsm kgxe2x88x921. With these solutions, recording pipettes have a resistance of 2-3 Mxcexa9. Using the whole-cell voltage clamp technique (Hamill et al.(1981)Pflxc3xcgers Arch., 391: 85-100), cells are voltage-clamped at xe2x88x9260 mV and control current responses to 1 mM glutamate are evoked. Responses to 1 mM glutamate are then determined in the presence of test compound. Compounds are deemed active in this test if, at a test concentration of 10 xcexcM, they produce a greater than 30% increase in the value of the current evoked by 1 mM glutamate.
In order to determine the potency of test compounds, the concentration of the test compound, both in the bathing solution and co-applied with glutamate, is increased in half log units until the maximum effect was seen. Data collected in this manner are fit to the Hill equation, yielding an EC50 value, indicative of the potency of the test compound. Reversibility of test compound activity is determined by assessing control glutamate 1 mM responses. Once the control responses to the glutamate challenge are re-established, the potentiation of these responses by 100 xcexcM cyclothiazide is determined by its inclusion in both the bathing solution and the glutamate-containing solution. In this manner, the efficacy of the test compound relative to that of cyclothiazide can be determined.
According to another aspect, the present invention provides a pharmaceutical composition, which comprises a compound of formula I or a pharmaceutically acceptable salt thereof as defined hereinabove and a pharmaceutically acceptable diluent or carrier.
The pharmaceutical compositions are prepared by known procedures using well-known and readily available ingredients. In making the compositions of the present invention, the active ingredient will usually be mixed with a carrier, or diluted by a carrier, or enclosed within a carrier, and may be in the form of a capsule, sachet, paper, or other container. When the carrier serves as a diluent, it may be a solid, semi-solid, or liquid material which acts as a vehicle, excipient, or medium for the active ingredient. The compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments containing, for example, up to 10% by weight of active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.
Some examples of suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum, acacia, calcium phosphate, alginates, tragcanth, gelatin, calcium silicate, micro-crystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, methyl cellulose, methyl and propyl hydroxybenzoates, talc, magnesium stearate, and mineral oil. The formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents, or flavoring agents. Compositions of the invention may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art.
The compositions are preferably formulated in a unit dosage form, each dosage containing from about 1 mg to about 500 mg, more preferably about 5 mg to about 300 mg (for example 25 mg) of the active ingredient. The term xe2x80x9cunit dosage formxe2x80x9d refers to a physically discrete unit suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical carrier, diluent, or excipient. The following formulation examples are illustrative only and are not intended to limit the scope of the invention in any way.
The above ingredients are mixed and filled into hard gelatin capsules in 460 mg quantities.
The active ingredient, starch, and cellulose are passed through a No. 45 mesh U.S. sieve and mixed thoroughly. The solution of polyvinylpyrrolidone is mixed with the resultant powders which are then passed through a No. 14 mesh U.S. sieve. The granules so produced are dried at 50xc2x0 C. and passed through a No. 18 mesh U.S. sieve. The sodium carboxymethyl starch, magnesium stearate, and talc, previously passed through a No. 60 mesh U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets each weighing 150 mg.
As used herein the term xe2x80x9cpatientxe2x80x9d refers to a mammal, such as a mouse, guinea pig, rat, dog or human. It is understood that the preferred patient is a human.
As used herein the term xe2x80x9ceffective amountxe2x80x9d refers to the amount or dose of the compound which provides the desired effect in the patient under diagnosis or treatment.
The particular dose of compound administered according to this invention will of course be determined by the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition being treated, and similar considerations. The compounds can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, or intranasal routes. Alternatively, the compound may be administered by continuous infusion. A typical daily dose will contain from about 0.01 mg/kg to about 100 mg/kg of the active compound of this invention. Preferably, daily doses will be about 0.05 mg/kg to about 50 mg/kg, more preferably from about 0.1 mg/kg to about 25 mg/kg.